Resolution of the current tuberculosis (TB) epidemic will require prevention of new TB infection as well as improved methods for treating existing disease. The long term objective of the proposed research program is to gain a better understanding, at the cellular and molecular levels, of how Mycobacterium tuberculosis (Mtb) establishes infection so that effective strategies can be developed to prevent it. This proposal focuses on the interaction of tubercle bacilli with host macrophages, a relationship that is central to the Mtb infection process. The specific aims of this project are to: I. address, by using histochemical and immunocytochemical techniques in conjunction with electron, fluorescence and brightfield microscopy, three questions about the Mtb-macrophage interaction. These questions are: a) does phagolysosome fusion induce Mtb gene expression? b) does the route of bacterial uptake by the cell affect the outcome of infection? and c) what determines bacterial localization within the cell? II. identify, using promoter fusions and 2-D gel electrophoresis, bacterial genes that are expressed specifically while in the macrophage. These genes are likely to be important in the pathogenesis of TB disease, and may provide useful diagnostic reagents. III. develop rapid fluorescence-based assays to facilitate the molecular and genetic study of bacterial genes which influence: a) phagolysosome fusion; b) bacterial uptake into host cells; and c) intracellular bacterial multiplication. IV. use fluorescence-based assays and genetic techniques to identify, clone and characterize additional bacterial genes that may play roles in TB pathogenesis. Genes of interest include those affect: a) bacterial localization within the cell; b) bacterial entry into host cells; and intracellular bacterial multiplication. This project will establish a foundation for future studies on the mechanism by which Mtb establishes infection. It will also facilitate the development of new TB diagnostics by vaccines.